Effect of Na + flux inhibitors on induction of c-fos, c-myc, and ODC genes during cell cycle

The role of Na + transport systems in the mitogenic signal induced by growth factors was studied, and it was shown that two Na + transport systems contribute to the early increase in cytoplasmic Na + in response to serum growth factors, namely the amiloride-sensitive Na+/H+ antiport and the bumetanide-sensitive Na+/K+/Cl- cotransport. Bumetanide or amiloride, when added separately, inhibited part of the increase in cytoplasmic Na +, as a response to the addition of serum to quiescent BALB/c mouse 3T3 fibroblasts. Each drug also suppressed part of the stimulation of the ouabain-sensitive Rb + influx, which was controlled by intracellular Na +. However, when both drugs were added together with serum growth factors, a complete inhibition of the early increase in [Na +], and subsequently a complete blockage of Na+/K+ pump stimulation was obtained. Amiloride or bumetanide, when added separately, only partially inhibited DNA synthesis induced by serum, 24% and 8% respectively. However, when both drugs were added together, at the time of serum addition to the quiescent cells, cell entry into S-phase was completely inhibited. To investigate the mode of cell-cycle inhibition, analysis was done of the possible role of early Na + fluxes in the mitogenic signal transduced from cell membrane receptors to the nucleus. The effects of the two drugs amiloride and bumetanide on induction of three genes--c-fos, c-myc, and ornithin decarboxylase (ODC)--was measured during cell transition through the G1-phase. Amiloride and bumetanide, when added separately or in combination, did not inhibit the induction of c-fos, c-myc, and ODC mRNAs. These results suggest that stimulation of Na + fluxes by serum growth factors is essential for cell transition into the S-phase of cell cycle, but it plays no apparent role in the growth factor signal transduced from the cell surface to the interior of the cell, as manifested by c-fos, c-myc, and ODC genes induction.     

Membrane potential and sodium flux in neuroblastoma X glioma hybrid cells: Effects of amiloride and serum

The Na⁺ uptake into neuroblastoma x glioma hybrid cells was measured in Hepes-buffered EMEM containing 10% calf serum and 5 mM ouabain in the presence and absence of amiloride (1.0 mM). Amiloride was found to markedly inhibit net Na⁺ influx (by approximately 50%). Examination of the effect of amiloride on net Na⁺ influx in the absence of calf serum revealed that a significant amiloride-sensitive Na⁺ influx remains even under serum-deprived conditions, although the degree of amiloride inhibition (35%) is substantially lower than that found in the presence of serum. The amiloride-insensitive portion of Na⁺ influx was found to be independent of serum effects. Estimation of resting membrane potential was made by measurement of the steady state distribution of the lipophilic cation, TPP⁺, in the presence and absence of amiloride. A large, immediate increase in TPP⁺ uptake, indicative of a membrane hyperpolarization, was seen upon addition of amiloride. Determination of the effect of amiloride on resting membrane potential of serum-deprived cells showed that cells are hyperpolarized to a greater extent in the presence than in the absense of amiloride, and that serum exerts a depolarizing effect on the cells. Thus, serum-stimulation of Na⁺ influx results in a depolarization of resting membrane potential, while amiloride inhibition of Na⁺ influx causes a hyperpolarization. These data strongly suggest that NG108-15 cells possess an electrogenic Na⁺ influx pathway that is sensitive to amiloride inhibition and enhanced by serum.     

Amiloride inhibits protein synthesis in isolated rat hepatocytes

The effects of amiloride on Na⁺ ion influx, amino acid transport, protein synthesis and RNA synthesis have been studied in isolated rat hepatocytes. The initial rate of ²²Na⁺ uptake and the amount of ²²Na⁺ taken up at later time points were decreased in hepatocytes incubated in the presence of amiloride. Amiloride inhibited by about 25% the influx of α-methylamino[1?¹⁴C]isobutyric acid, a specific substrate for the A (Alanine preferring) system of neutral amino acid transport. By contrast, the activity of system L (Leucine preferring) was not affected by amiloride. Rates of protein synthesis were determined by using high extracellular concentrations of [¹⁴C]valine in order to maintain a constant amino acid precursor pool. Amiloride inhibited protein synthesis by 85% and had no effect on RNA synthesis. Half-maximal inhibition of protein synthesis occurred with amiloride at about 150 μM. In the absence of Na⁺ in the incubation medium, the rate of protein synthesis was reduced by about 35% and no further inhibition was observed with amiloride. These results suggest that in isolated rat hepatocytes protein synthesis is partially dependent on Na⁺, and that amiloride is an efficient inhibitor of protein synthesis.     

Isolation and characterization of an amiloride-resistant mutant of Methanothermobacter thermautotrophicus possessing a defective Na+/H+ antiport

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant to the Na+/H+ antiporter inhibitor amiloride was isolated. The Na+/H+ exchanger activity in the mutant cells was remarkably decreased in comparison with wild-type cells. Methanogenesis rates in the mutant strain were higher than wild-type cells and resistant to the inhibitory effect of 2 mM amiloride. In contrast, methanogenesis in wild-type cells was completely inhibited by the same amiloride concentration. ATP synthesis driven by methanogenic electron transport or by an electrogenic potassium efflux in the presence of sodium ions was significantly enhanced in the mutant cells. ATP synthesis driven by potassium diffusion potential was profoundly inhibited in wild-type cells by the presence of uncoupler 3,3',4',5- tetrachlorosalicylanilide and sodium ions, whereas c. 50% inhibition was observed in the mutant cells under the same conditions.     

Effect of sodium, lithium and amiloride on the K+-dependent phosphatase activity of membrane preparations of Na,K-ATPase

Effects of sodium, lithium and amiloride on the ATPase reaction and on its potassium-dependent step were studied using membrane preparations of Na,K-ATPase. It was established that the addition of 70 mM NaCl or LiCl to the reaction medium diminished the hydrolysis of para-nitrophenyl phosphate (pNPP) by 70 and 40%, respectively. Amiloride (0.8 mM) inhibited activities of Na,K-ATPase and pNPPase by 50 and 15%, respectively. The higher concentrations of amiloride produced a more prominent inhibition of Na,K-ATPase, but not of pNPPase. There was no correlation between the effect of amiloride on the pNPP hydrolysis and potassium concentration in the medium. There was the additivity in the inhibition of pNPPase by 0.8 mM amiloride and sodium or lithium ions up to the concentrations of ions as high as 30 mM. A conclusion is made that the inhibition of Na,K-ATPase by amiloride is mediated through the modification of the sensitivity of the enzyme to sodium.     

The effect of amiloride on hormonal regulation of amino acid transport in isolated and cultured adult rat hepatocytes

Insulin and glucagon stimulate amino acid transport in isolated rat hepatocytes. Amiloride, a specific Na+-influx inhibitor, completely inhibited the hormonal (glucagon or insulin) stimulation of alpha-aminoisobutyric acid influx by preventing the emergence of a high-affinity transport component. The drug also inhibited [14C]valine incorporation into hepatocyte protein. The half-maximal concentration of amiloride for inhibition of protein synthesis was similar to that required for inhibition of hormone-stimulated amino acid transport (approx. 0.1 mM). In primary cultured rat hepatocytes, amiloride markedly depressed the stimulation of alpha-aminoisobutyric acid transport by glucagon, or a mixture of glucagon, insulin and epidermal growth factor. These results suggest that amiloride inhibits the hormonal stimulation of hepatocyte amino acid transport by preventing the synthesis of high-affinity transport proteins. They also suggest that the hormonal stimulation of hepatocyte amino acid transport is dependent, at least partly, on Na+ influx.     

Characteristics of an amiloride-sensitive sodium entry pathway in cultured rodent glial and neuroblastoma cells

We have studied the induction of an amiloride-sensitive sodium influx into C6 glioma, NIE, and NB2A neuroblastoma cell lines. In late log phase, cells grown continuously in the presence of 10% fetal calf serum showed Na⁺ influxes of approximately 25–30 nmol/mg protein min; < 5% of this flux was inhibited by amiloride. Removal of serum for 24 h caused a decrease in the total Na⁺ influx to 15–20 nmol/mg protein/min. Upon readdition of serum to the incubation medium, there was an increase in total Na⁺ influx, depending on the cell type, of 20–400% within 2 min. This increment in Na⁺ influx represented an increase in amiloride-sensitive Na⁺ transport with an apparent K′, of 0.4 mM. By adding serum back at various times after serum deprivation, it was determined that 4 h was required to observe a detectable increase in the amiloride-sensitive Na⁺ flux. Thus, serum removal results in the induction of the amiloride transport system which, however, remains latent until the reintroduction of serum to the medium. Addition of 5 μg/ml of cycloheximide blocked the increase in Na⁺ transport, indicating that de novo protein synthesis mediated this serum deprivation–induced increase in Na⁺ transport. Moreover, inhibition of de novo lipid synthesis by 0.1 mM fenfluramine also blocked the induction of this transport activity, suggesting that a coordinated synthesis of lipid and protein is required for the expression of this sodium transport site. We have also found that this serum stimulated Na⁺ influx did not saturate with Na⁺ concentration, up to 140 mM. Also, among commonly used inhibitors of passive Na⁺ entry into epithelial tissues, only amiloride was capable of inhibiting this transport system in these neural cell lines.     

Amiloride inhibits protein synthesis and lowers the intracellular pH in exponential growing Yoshida rat ascites hepatoma (AH 130) cells: evidence for a role of the Na+/H+ exchanger

We have previously demonstrated in a rat ascites hepatoma cell line (Yoshida AH 130) the presence of a glucose-activatable and amiloride sensitive Na+/H+ exchange (Cell Biol. Int. Rep., 1984, 8, 297-307). Amiloride is known to inhibit this exchange and to cause a cytoplasmic acidification, with inhibition of protein and DNA synthesis, in cells induced to grow. Amiloride appears also to penetrate the cells and to inhibit directly protein synthesis. In the present report we describe experiments in which the activity of amiloride (0.1, 0.4 and 3.0 mM) on protein synthesis and the internal pH of cells was compared in exponential growing and stationary phase Yoshida ascites cells. In phosphate buffered medium and Na+ out = 147 mM no inhibition of protein synthesis (3H-leu incorporation into total cell protein) and no internal acidification (14C-DMO distribution between intra- and extracellular volume) were produced by 0.1 and 0.4 mM amiloride in exponential growing cells. In stationary phase cells, on the contrary, 0.4 mM amiloride inhibited protein synthesis by 60% without decreasing the internal pH. When the Na+ out was lowered to 25 mM, to reduce competition with amiloride, and/or all Na+ out was substituted with choline, 0.1 and 0.4 mM amiloride markedly inhibited protein synthesis and decreased the internal pH in exponential growing cells. No apparent inhibition occurred in stationary phase cells under the same conditions, possibly due to a preexistent internal acidification, with severe decrease of protein synthesis. Fluorimetric studies of amiloride "binding" to ascites cells showed that a reduced number of amiloride receptor sites could exist in Yoshida hepatoma cells at the stationary phase of growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of potent Na+/H+ exchange inhibitors from the amiloride series in A431 cells

Na+/H+ exchange is stimulated in a variety of cell types by addition of mitogenic polypeptides such as epidermal growth factor or platelet-derived growth factor. In order to assess the importance of Na+/H+ exchange in the mitogenic response, it is desirable to have available inhibitors of this process which exhibit high affinity and good specificity. We characterize in this report a number of 5-alkylamino-substituted derivatives of amiloride [3,5-diamino-6-chloro-N-(diaminomethylene)pyrazinecarboxamide++ +] which show much higher affinity than the parent compound for the Na+/H+ antiporter in A431 cells. High affinity is conferred by substitution with two alkyl groups and is increased by introducing a branched alkyl chain. An analogue bearing a 5-anilino group is also very potent. These analogues effectively inhibit the elevation of intracellular pH upon stimulation of Na+/H+ exchange by growth factors. We have assessed other potential inhibitory effects of these compounds on cellular metabolism. In agreement with previous reports, we find that amiloride inhibits protein synthesis both in cells and in cell-free translation systems. While amiloride and its analogues show similar inhibition of protein synthesis in a cell-free system, most analogues inhibit cellular protein synthesis at much lower concentrations than does amiloride. These analogues are also potent inhibitors of purified Na,K-ATPase and cause a profound decrease in intracellular K+ as well as ATP content. These latter effects, however, require analogue concentrations which are 5-7 times higher than those inhibiting cellular protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of amiloride on the induction of DNA synthesis and casein gene expression in rabbit mammary explants

Amiloride, an inhibitor of Na+/H+ exchange, was added at various concentrations to the culture medium of rabbit mammary explants. In the concentration range 100-250 microM, amiloride progessively inhibited 14C-thymidine incorporation induced by insulin, EGF or prolactin. Up to 250 microM, amiloride, which did not inhibit basal protein synthesis, was not cytotoxic, but it reduced basal DNA synthesis at the highest concentration. Addition of amiloride to the culture medium of mammary explants also strongly inhibited the induction of casein synthesis and casein mRNA accumulation by prolactin. The inhibition by amiloride is therefore not specific of the mitogenic action of prolactin since this drug also prevented its lactogenic action. The data reported here describe a new inhibitory action of amiloride on the transmission of the lactogenic signals.     

A dextran-bound amiloride derivative is a selective inhibitor of Na+/H+ antiport. Application for studying the role of the antiporter in cellular proliferation in human fibroblasts

Inhibitors of Na+/H+ exchange from the amiloride series are known to accumulate within the cell and cause an inhibition of a variety of cellular functions. In order to render the amiloride molecule impermeable to cells, we have synthesized a potent amiloride analog, 5-N-(3-aminophenyl)amiloride (compound A35, Ki = 60 nM). The isothiocyanate derivative of A35 (A35-NCS) was coupled to soluble dextrans of 15-20 kDa that have been derivatized with diaminoalkane spacer groups. Dextran-bound amiloride derivatives showed good inhibition of Na+/H+ exchange in human foreskin fibroblasts and A431 cells. Among several spacer groups tested, dextran derivatized with ethylenediamine showed the highest inhibitory activity. The intrinsic inhibitory potency of this polymer increased with increasing degree of substitution with A35, approaching that of free A35 with substitution of approximately 3 mol of A35 per mole of dextran. Coupling to dextran largely diminished side effects of the amiloride derivative on cells such as the inhibition of protein synthesis. A35-dextran was an effective inhibitor of serum-induced reinitiation of DNA synthesis in human foreskin fibroblasts in a bicarbonate-free medium, pH 7.1, but had little effect when either the pH of the medium was more alkaline or when the medium contained a bicarbonate buffer. These findings suggest that the selective inhibition of Na+/H+ antiport by A35-dextran prevents the reinitiation of DNA synthesis when the external conditions are such that the antiporter activity is required for the establishment of a permissive intracellular pH. Polymer-bound amiloride analogs should be useful as selective inhibitors in studies of the physiological role of the Na+/H+ antiporter, as well as for affinity purification of the antiporter.     

Phospholipid metabolism and T cell activation: receptor triggering is associated with the inhibition of phosphatidylserine synthesis

Activation of Jurkat T lymphocytes to produce IL2 is accompanied by a strong inhibition of phosphatidylserine (PS) synthesis. This inhibition was obtained either with the mitogenic lectin PHA, anti-CD3 monoclonal antibodies (mAb), anti-CD2 mAb or anti-Ti mAb. Bypassing membrane receptor signalling, by using a Ca2+ ionophore or a protein phosphatase inhibitor, sodium ortho-vanadate, also results in a marked inhibition of PS synthesis. Activators of phospholipid -Ca2+ dependent protein kinase C (PKC) did not significantly modify PS synthesis, suggesting that the observed changes only involve the transduction of the first activation signal. PS being a necessary cofactor for PKC, our results strongly suggest that the inhibition of PS synthesis induced by receptor triggering exerts a feed back control on PKC therefore leading to a transient activation of the enzyme upon full lymphocyte activation.     

Mitogenesis of 3T3 fibroblasts induced by endogenous ganglioside is not mediated by cAMP, protein kinase C, or phosphoinositides turnover

The B subunit of cholera toxin, which binds specifically to ganglioside GM1, stimulates DNA synthesis in quiescent Swiss 3T3 fibroblasts grown in chemically defined medium. The mitogenic response to the B subunit was potentiated by insulin and other growth factors. To elucidate the mechanism by which the B subunit stimulates cell growth , its effects on several transmembrane signaling systems which have been suggested to play a vital role in cell growth regulation were examined. The B subunit did not increase cAMP levels nor activate adenylate cyclase. The B subunit induced a rapid and profound increase in intracellular free Ca2+ as measured with the fluorescent Ca2+-sensitive dye quin 2/AM. Removal of external Ca2+ completely inhibited the signal, thus suggesting that the B subunit elevates intracellular Ca2+ through a net influx of extracellular Ca2+ rather than by causing the release of Ca2+ from intracellular stores. These findings are consistent with the observations that the B subunit induced reinitiation of DNA synthesis without activation of phospholipase C. There was no increase in the formation of inositol trisphosphate, the second messenger that mediates release of Ca2+ from intracellular stores. In addition, the B subunit still stimulated DNA synthesis in Swiss 3T3 cells pretreated with phorbol ester to down-regulate protein kinase C. These results suggest that the mitogenic effects of the B subunit are mediated mainly by facilitation of Ca2+ influx and that activations of adenylate cyclase, phospholipase C, or protein kinase C are not obligatory steps in the initiation of cell growth by the B subunit. Furthermore, the observation that Ca2+ ionophores, such as ionomycin and A23187, are not mitogenic implies that additional undefined growth signaling pathways may exist in this system.     

The ATP4- receptor-operated ion channel of human lymphocytes: inhibition of ion fluxes by amiloride analogs and by extracellular sodium ions.

Extracellular ATP is known to increase the membrane permeability of a variety of cells. Addition of ATP to human leukemic lymphocytes loaded with the Ca2+ indicator, fura-2, induced a rise in cytosolic Ca2+ concentration which was attenuated or absent in NaCl media compared with KCl, choline Cl, or NMG Cl media. In contrast, anti-immunoglobulin antibody gave similar Ca2+ transients in NaCl and KCl media. A half-maximal inhibition of peak ATP-induced Ca2+ response was observed at 10-16 mM extracellular Na+. Basal 45Ca2+ influx into lymphocytes was stimulated 9.6-fold by ATP added to cells in KCl media, but the effect of ATP was greatly reduced for cells in NaCl media. Hexamethylene amiloride blocked 74% of the ATP-stimulated Ca45 uptake of cells in KCl media. Flow cytometry measurements of fluo-3-loaded cells confirmed that the ATP-induced rise in cytosolic Ca2+ was inhibited either by extracellular Na+ or by addition of hexamethylene amiloride. Extracellular ATP stimulated 86Rb efflux from lymphocytes 10-fold and this increment was inhibited by the amiloride analogs in a rank order of potency 5-(N-methyl-N-isobutyl)amiloride greater than 5-(N,N-hexamethylene)amiloride greater than 5-(N-ethyl-N-isopropyl)amiloride greater than amiloride. ATP-induced 86Rb efflux showed a sigmoid dependence on the concentration of ATP and Hill analysis gave K1/2 of 90 and 130 microM and n values of 2.5 and 2.5 for KCl and NaCl media, respectively. However, the maximal ATP-induced 86Rb efflux was 3-fold greater in KCl than in NaCl media. Raising extracellular Na+ from 10 to 100 mM increased ATP-induced Na+ influx from a mean of 2.0 to 3.7 nEq/10(7) cells/min, suggesting either saturability or self-inhibition by Na+ of its own influx. These data suggest that ATP opens a receptor-operated ion channel which allows increased Ca2+ and Na+ influx and Rb+ efflux and these fluxes are inhibited by extracellular Na+ ions as well as by the amiloride analogs.     

Amiloride inhibition of DNA synthesis and immunoglobulin production by activated human peripheral blood mononuclear cells is independent of sodium/hydrogen antiport

Activation of sodium/proton (Na+/H+) antiport activity has been shown to occur as an early event in mitogenesis. Because amiloride inhibits Na+/H+ antiport activity, it is hypothesized that mitogenesis may be inhibited by amiloride. In this work, we examined the effect of amiloride on DNA synthesis as measured by [3H]thymidine uptake and immunoglobulin (Ig) production as measured by an ELISA system in human peripheral blood mononuclear cells (PBM). Amiloride at 100 microM concentration inhibited irradiated Raji cell (*R)-activated and phytohemagglutinin-P (PHA-P)-stimulated DNA synthesis by 50 +/- 11% and 72 +/- 12%, respectively. IgG production was inhibited by 71% at 100 microM amiloride concentration in *R-activated PBM. This concentration of amiloride inhibited Na+/H+ antiport activity by 92%. Because amiloride is known to inhibit other pre-replicative cellular functions such as protein synthesis, we used an amiloride analogue, dimethylamiloride, which inhibited Na+/H+ antiport activity by 90% at a concentration of 1 microM without inhibition of PBM Ig or DNA synthesis. Furthermore, neither PHA-P nor *R-stimulated PBM demonstrated an intracellular alkalinization even after 6 hr of stimulation. Similarly, T cell-enriched or B cell-enriched populations did not show intracellular alkalinization after PHA-P or *R activation. Thus, it appears that Na+/H+ antiport activation is not an early event in PBM mitogenesis. The inhibition of mitogenesis by amiloride may be due to abrogation of premitotic events such as protein synthesis.     

Serum stimulation of potassium fluxes,ouabain binding,and sodium fluxes in quiescent chicken embryo fibroblasts

Growth-contingent alterations in potassium and sodium fluxes, ouabain binding, and potassium ion content were examined following serum stimulation of quiescent, density-inhibited chicken embryo fibroblasts. Serum stimulation resulted in very rapid 1.5- to 1.8-fold increases in ouabain-sensitive potassium influx and lesser 1.4- to 1.5-fold increases in potassium efflux and sodium influx. Potassium influx stimulation was maximal after addition of 5–20% calf serum and was unaffected by cycloheximide inhibition of protein synthesis. Reflecting the slightly greater stimulation of potassium influx versus potassium efflux, potassium ion levels were 10–15% higher in serum-stimulated compared to unstimulated cells. Specific ouabain binding levels in stimulated and unstimulated control cells were initially similar, however, by four hours after stimulation a 40–50% increase in specific ouabain binding was observed. Incubation with ouabain was found also to inhibit later serum-stimulated hexose uptake and thymidine incorporation; this blockage may be a consequence of subnormal potassium levels rather than ouabain inhibition of the serum-stimulated potassium influx.     

Demonstration of a late amiloride-sensitive event as a necessary step in initiation of DNA synthesis by thrombin

Amiloride, a Na⁺ influx inhibitor, has been shown to inhibit initiation of DNA synthesis by thrombin in mouse embryo fibroblast-like cells. Long exoosures (24 hr) to high concentrations of amiloride inhibited incorporation of thymidine into the DNA of both thrombin-stimulated and nonstimulated cells, suggesting that this inhibition might not be specific for thrombin-initiated DNA synthesis. Fluorescence microscopy and spectrofluorimetry showed that amiloride was internalized with an apparent mitochondrial association and that the internalized amiloride was readily released from the cells after removing amiloride from the medium. Based on this reversibility, cells were exposed to amiloride for short periods of time during thrombin treatment to determine the temporal relationship between any amiloride-sensitive event(s) and initiation of DNA synthesis. The presence of amiloride (100 μM) during a 12-hr exposure to thrombin did not block thrombin-initiated DNA synthesis or cell division but did delay the onset of DNA synthesis and the peak of thymidine incorporation into DNA by approximately 3 hr, suggesting that early initiation events might proceed in the presence of amiloride. ⁸⁶Rb⁺ transport studies demonstrated that in this system ouabain-sensitive K⁺ uptake via the Na, K-ATPase was stimulated by thrombin during both an early and a late period. This stimulation was amiloride-sensitive under the same conditions used for growth experiments, suggesting that amiloride was inhibiting thrombin-stimulated Na⁺ transport in this system. Additional experiments showed that exposing cells to amiloride only during the first 8 hr after thrombin addition did not inhibit initiation. The presence of amiloride from 8–12 hr after thrombin addition maximally inhibited thrombin-stimulated DNA synthesis. Together these results demonstrate that amiloride inhibits thrombin-initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8–12 hr after thrombin addition.     

Sodium-hydrogen exchange system in brush border membranes from cortical and medullary regions of the proximal tubule

The Na+/H+ exchange system was studied in brush border membrane vesicles isolated from cortical and medullary regions of the proximal tubule of rabbit kidney. The activity of the exchanger was assessed by measuring hydrogen influx (monitored by acridine orange fluorescence), 22 Na influx and the sensitivity of these fluxes to amiloride and its analogue ethylisopropyl amiloride. In contrast to previously published data (indicating the absence of pH-gradient driven and amiloride sensitive 22Na-influx in medullary site vesicles (13, 15], Na+/H+ exchange activity could be detected in both membrane preparations by sodium tracer and fluorescence detection of hydrogen influx. Amiloride inhibition of 22Na influx was more effectively protected by increasing sodium concentration in cortical than in medullary vesicles, suggesting differences in the action of amiloride in these preparations.     

Activation of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange by protein phosphatase inhibitors in red blood cells of the frog<Emphasis Type="Italic"> Rana ridibunda</Emphasis>

The present study was designed to evaluate the role of protein phosphatases in regulation of sodium transport in the marsh frog erythrocytes using 22Na as a tracer. For this purpose the cells were treated with several known inhibitors of protein phosphatases. In standard isotonic medium, exposure of the cells to 10 mmol l(-1) NaF, 20 nmol l(-1) calyculin A or 0.1 mmol l(-1) cantharidin resulted in a significant (1.7-fold) increase in unidirectional ouabain-insensitive Na+ influx. The Na+ influx in frog red cells was progressively activated as the medium osmolality was increased by addition of 100, 200 or 300 mmol l(-1) sucrose to standard isotonic medium. The stimulatory effect of protein phosphatase blockers on Na+ influx was much higher in hypertonic medium containing 100 or 200 mmol l(-1) sucrose than that in isotonic medium. Stimulation of Na+ transport enhanced with increasing concentrations of calyculin A, and half-maximal activation (EC50) was obtained at 16 nmol l(-1). However, Na+ influx induced by strong hypertonic treatment (+300 mmol l(-1) sucrose) was not altered further in the presence of protein phosphatase inhibitors. The changes in Na+ influx evoked by protein phosphatase inhibitors and hypertonic treatment were associated with a rise in the intracellular Na+, but not K+, content. Enhancement in Na+ influx after addition of protein phosphatase blockers to cell suspension in isotonic or hypertonic media was almost completely inhibited by Na+/H+ exchange inhibitors, amiloride and ethyl-isopropyl-amiloride. The basal Na+ influx in frog erythrocytes in isotonic medium was relatively low (1.7 mmol/l cells/h) and not affected by 1 mmol l(-1) amiloride. Thus, the data obtained clearly indicate that Na+/H+ exchanger in the marsh frog red blood cells is under tight regulatory control, in all likelihood via protein phosphatases of types PP-1 and PP-2A.     

Effects of amiloride on the transport of sodium and other ions in the alga Hydrodictyon reticulatum

The diuretic amiloride, an almost specific inhibitor of sodium transport in animal cells and tissues, appears to produce a number of effects in the alga Hydrodictyon reticulatum. At 1 mmol/l concentration it markedly reduces the influx of sodium ions (but not their active outflux), the influxes of potassium, chloride as well as of bicarbonate ions, and causes a profound decrease in the plasmalemma membrane potential. This plurality of inhibitory effects suggests that individual transport processes in the alga are mutually coupled.